The first genome assembly of the amphibian nematode parasite (Aplectana chamaeleonis)

Cosmocercoid nematodes are common parasites of the digestive tract of amphibians. Genomic resources are important for understanding the evolution of a species and the molecular mechanisms of parasite adaptation. So far, no genome resource of Cosmocercoid has been reported. In 2020, a massive Cosmocercoid infection was found in the small intestine of a toad, causing severe intestinal blockage. We morphologically identified this parasite as A. chamaeleonis. Here, we report the first A. chamaeleonis genome with a genome size of 1.04 Gb. The repeat content of this A. chamaeleonis genome is 72.45%, and the total length is 751 Mb. This resource is fundamental for understanding the evolution of Cosmocercoid and provides the molecular basis for Cosmocercoid infection and control.


INTRODUCTION
Amphibians are widely distributed across the world, with a high diversity of species. More than 6,000 amphibian species exist worldwide, significantly contributing to the global biodiversity (http://www.amphibiachina.org/). Nematodes can interfere with the fitness of their amphibian hosts and ultimately affect their survival [1]. They can also facilitate adaptions in their host physiology, acting as a major force to promote their coevolution.
Parasitic infections are highly prevalent in amphibians, but few systematic studies have been conducted on this topic [2]. The order Ascaridomorpha, a typical representative of large parasitic nematodes [3], contains five families: Ascaridoidea, Cosmocercoidea, Heterakoidea, Seuratoidea and Subuluroidea. Only Ascaridoidea has genomic data, and most of its genera have mammalian hosts. The genomic data of amphibian parasites from all five families are deficient. Within Ascaridomorpha, Cosmocercoidea is the most abundant family of amphibian roundworms [4]. The genus Aplectana (Cosmocercoidea: Cosmocercidae), which includes Aplectana hylae [5], Aplectana macintoshii, Aplectana hainanensis [6], Aplectana paucipapillosa [6], Aplectana xishuangbannaensis [7] and Aplectana chamaeleonis [1], includes common parasites of amphibians. Both nematodes and amphibians are important components of the ecosystem. The lack of molecular resources for amphibian parasites made it impossible to study their adaptability and evolutionary history, thus hindering the development of related fields. At present, research on Aplectana mainly focuses on its morphological identification. However, this genus is difficult to distinguish only through morphology, as this parameter is affected by the developmental time and individual differences. Therefore, understanding the genus Aplectana simply through morphological identification is not enough.
In the present study, we report the first highly complete A. chamaeleonis genome, with a genome size of 1.04 Gb. Its repeat element content reaches 72.45%, providing new evidence for understanding the relationship between repeat elements and genome size in Ascaridomorpha species. Furthermore, as this is the first genome of a Cosmocercidae species, it enhances our understanding of the evolution of Cosmocercidae species and their adaptive molecular mechanisms to amphibian hosts.

Context
We present a highly-complete genome assembly of A. chamaeleonis (Figure 1, NCBI:txid2696335), providing a valuable resource for evolutionary biology, ecology and phylogenetics. The genome size is 1.04 Gb ( Table 1). The average sequence length is 496 kb, and the N50 length is 1.08 Mb. The maximum length is 8.07 Mb, and the minimum is 34 kb.
Our A. chamaeleonis genome has a GC content of 45%, and its N50 is longer than in other nematodes at the scaffold level while it is relatively shorter than in some of them at the chromosome scale. Additionally, the integrity of the genome was assessed at 76.9% using Benchmarking Universal Single-Copy Orthologs (BUSCO, RRID:SCR_015008). The characteristics of the genome sequence showed that the genome is large and has high integrity. Blobtools (RRID:SCR_017618) was used for genomic quality control and taxonomic partitioning. The results showed that 91% of the sequences aligned to Nematoda (1898/2088) and 7% to Arthropoda (122/2088). This will be an invaluable resource for understanding amphibian parasites.
According to the reported total repeat length and proportion of the Ascaridomorpha species, the content of the repeat elements in nematodes varies significantly, ranging from 3.92% to 45.25% [8]; in species with larger genomes, the content of repeat elements occupies a large proportion. There were significant differences in the total length of the genome and proportion of repeat content among different nematodes, which varied from 87 Mb to 751 Mb and from 8.34% to 72.45%, respectively (Table 1) [9]. In A. chamaeleonis, the total length of the genome is 1,036,852,746 bp, and the content of repetitive elements in the genome reaches a staggering 72.45%, with a total length of 751 Mb (Tables 1-3). We counted the content of the various repeating elements: unknown types of repeating elements account for 51%, whereas LINE and DNA account for 10% and 8%, respectively ( Figure 2). These findings confirm that the large number of repeated sequences is one of the leading causes for such a large genome.
A total of 12,887 functional genes were annotated. Additionally, all genes were annotated with KEGG (RRID:SCR_012773) and found to be primarily represented in pathways such as    According to the demographic history scale of A. chamaeleonis (Figure 4), the population size of A. chamaeleonis gradually increased between 200,000 and 100,000 years ago. Then, during the last glacial maximum, the population of A. chamaeleonis gradually decreased, which may be linked to the decline of its host population during the same period [10].
The infection rate of the Cosmocercidae species in amphibians is high, but no genome information is available for any Cosmocercidae species. The genomes of A. chamaeleonis assembled in this study are an important resource for studying amphibian parasites. In particular, it may improve our understanding of the evolution of amphibian parasites and the molecular basis of the genome for adapting to amphibian hosts.

Sample collection and sequencing
A. chamaeleonis was collected from a Bufo pageoti infected with A. chamaeleonis in Shenzhen, China. All samples were thoroughly cleaned with sterile physiological saline (37°C), quickly frozen, transported on dry ice, and kept at −80 °C until further use. By using the microscope, morphological identification was carried out (Olympus). All experimental designs and nematode handling were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University. Sodium dodecyl sulphate/proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation were used to isolate whole genomic DNA [11]. The DNA quantity was estimated using a Qubit fluorometer with the dsDNA high-sensitivity kit (Invitrogen) and using agarose gel (1.0%) electrophoresis.
Genomic DNA was purified for long-read library preparation according to the manufacturer's instructions of the Nanopore platform, followed by long-read sequencing.

Genome assembly, duplicate purging
The Nanopore long reads were assembled with NextDenovo software (v2.0-beta.1; https://github.com/Nextomics/NextDenovo). Then NextPolish (v1.0.5) [12] was used to conduct a second round of correction and a third round of polishing for this assembly using the Whole Genome Sequencing (WGS) data. We used diamond (v0.9.10; RRID:SCR_016071) to blast the genome against the NCBI Non-Redundant Protein Sequence Database (NR) database. We then deleted the scaffolds that blasted to bacteria (such as Escherichia coli and Lactococcus lactis) and generated the clean genome. To get a haploid representation of the genome, duplicates were purged from the genome using the Purge_Dups pipeline (RRID:SCR_021173) [13]. To evaluate the quality of the genomes, a new software called blobtools was used for genomic quality control and taxonomic partitioning. The completeness of the genome was evaluated using the sets of BUSCO (v5.2.2) with genome mode and lineage data from nematode odb9 and eukaryote odb9, respectively [14].

Demographic history of A. chamaeleonis
We inferred the demographic history of A. chamaeleonis. The generation we used was 0.17 years per generation, and the mutation rate was 9 × 10 −9 single nucleotide mutations per site per generation on average [8]. We also used 100 bootstrap replicates to estimate the demographic history.

DATA AVAILABILITY
The data that support the findings of this study have been deposited into the CNGB Sequence Archive (CNSA) [25] of the China National GeneBank Database (CNGBdb) [26] with the accession number CNP0003496, and in NCBI under the biosample number PRJNA895947. Additional data is also available in the GigaDB repository [27].